2.1. Chimeric and/or Hybrid Duplex Nucleic Acids
The field of the invention concerns nucleic acids. Nucleic acids are heteropolymers, i.e., polymers of non-identical subunits, which are linked by oriented phosphodiester bonds or their derivatives, into polymers. Duplex nucleic acids are nucleic acids wherein each base of a first strand of the duplex corresponds to a base of a second strand of the duplex according to the scheme in which uracil or thymine and adenine correspond and cytosine and guanine correspond. Anti-parallel duplex strands having these correspondences are said to be Watson-Crick paired. Duplex nucleic acids can be of two major types, ribonucleic acids and deoxyribonucleic acids. Each ribonucleotide has an equivalent deoxyribonucleotide, e.g., adenosine and deoxyadenosine, cytidine and deoxycytidine, guanosine and deoxyguanosine, uridine and thymidine. As used in the field, a nucleic acid in which both ribonucleotides and deoxyribonucleotides are present in the same strand is termed a mixed or chimeric (hereinafter "chimeric") nucleic acid. A duplex nucleic acid in which deoxyribonucleotides and ribonucleotides correspond with each other is termed a hybrid-duplex. When two strands form a region of duplex nucleic acid for less than all of their bases, the resultant molecule is termed a heteroduplex.
Most often, the two strands of a duplex nucleic acid are not covalently bonded but are associated only by Watson-Crick pairing. However, the two strands of a duplex can be linked by an oligonucleotide to form a single polymer. The linking oligonucleotide is not Watson-Crick paired. The heteroduplex in which the first and second strands are portions of a single polymer is termed a "hairpin duplex" or a "stem and loop" structure. The former term will be used hereinafter.
As used herein chimerism is a property of a nucleic acid polymer and hybridism is a property of a duplex. For example, an mRNA and its template form a hybrid duplex though neither is chimeric, while, for example, the chimeric octanucleotides 5'd(TTTT)-r(CCCC)3' and 5'r(GGGG)-d(AAAA)3' will form a Watson-Crick duplex with each other but the resultant duplex is not a hybrid-duplex. A duplex nucleic acid which is not a hybrid-duplex is termed hereinafter a "homo-duplex". Unless specifically noted otherwise, a homo-duplex nucleic acid refers only to a deoxynucleotide containing duplex. Lastly, note that while those in the field refer to the formation of a Watson-Crick duplex as "hybridization," even where there is no hybrid-duplex nucleic acids.
Those interested in the study the structure of chimeric and/or hybrid duplex nucleic acids by X-ray diffraction and 2-dimensional NMR have synthesized chimeric nucleic acids and hybrid duplex nucleic acids for use in their studies. See, e.g., Salazar, M., et al., 1994, J.Mol.Biol. 241:440-55 and Egli, M., et al., 1993, Biochemistry 32:3221-37 (two stranded chimeric hybrid duplex of the form r.sub.3 d.sub.7 .multidot.d.sub.10); Ban, C., et al., 1994, J.Mol.Biol. 236:275-85 (self complementary chimeric hybrid duplex of the form d.sub.5 r.sub.1 d.sub.4); Chou, S. H., 1991, Biochemistry 30:5248-57 (self-complementing and non-self-complementing chimeric hybrid duplexes of the form d.sub.4 r.sub.4 d.sub.4). The complementary strands of these duplex nucleic acids were not covalently bound to each other; they were associated only by Watson-Crick pairing.
A second group of scientists who have synthesized chimeric nucleic acids are those interested in the study and use of ribozymes, i.e., RNA molecules that are either self-cleaving or cleave other RNAs. Perreault, J. P., et al., 1990, Nature 344:565; Taylor, N. R., et al., 1992, Nucleic Acids Research 20:4559-65; Shimaya, T., 1993, Nucleic Acids Research 21:2605. These researches have found that chimeric ribozymes are active and are more resistant to nuclease digestion than RNA ribozymes. Chimeric ribozymes are self-complementary, i.e., the Watson-Crick paired strands are covalently linked. The compounds synthesized during the studies of chimeric ribozymes differ from the above-noted hybrid-duplex molecules, that were synthesized used for structural studies, in that chimeric ribozymes do not contain stable hybrid-duplexes. Thus, a chimeric ribozyme having DNA binding arms binds to its substrate and forms a hybrid duplex. Yang, J. H., et al., 1990, Biochemistry 29:11156-60. See also, Sawata, S., et al., 1993, Nucleic Acids Research 21:5656-60; Hendry, P., et al., 1992, Nucleic Acids Research 20:5737-41 Shimayama, T., 1993, Nucleic Acids Research 21:2605. The ribozyme catalyzes the cleavage of the RNA substrate and the hybrid-duplex is thus dissolved.
2.2. Site-Directed Genetic Alteration in Eukaryotic Cells
Those skilled in the art of molecular biology recognize that on frequent occasions it is desired not merely to introduce a new polynucleic acid sequence, i.e, a new gene, into a targeted eukaryotic cell, but rather to alter a defined, pre-existing gene in the targeted cell. The targeted cell can be used in culture or it can be used to construct a transgenic animal.
A wide variety of techniques have been developed to introduce DNA into cultured eukaryotic cells. These techniques include calcium phosphate precipitation and DEAE-dextran mediated endocytosis, electroporation, liposome mediated fusion and transduction with replication incompetent viruses. However, while such techniques can quite often introduce functional genes into the eukaryotic cell, these techniques do not readily accomplish an alteration (mutation) in a specific existing gene. After introduction the exogenous DNA is isolated at a random position in the cell's genome by illegitimate recombination, rather than at a specific position by homologous recombination.
To date there is no generally satisfactory scheme for introducing a site-directed or site-specific genetic alteration (mutagenesis) in a higher eukaryote, i.e, in mammalian or avian cells. Although homologous recombination can be obtained in higher eukaryotic cells by introduction of very long (&gt;1 kb) nucleic acids, these techniques require the application of elaborate selection techniques because the rate of illegitimate recombination in higher eukaryotes greatly exceeds that of homologous recombination. Thomas, K. R. & Capecchi, M. R., 1987, Cell 52:503. See, also, Valancius, V. & Smithies O., 1991, Mol. Cell. Biol. 11:4389 (comparison homologous recombination of linearized and supercoiled plasmids in eukaryotic cells).
One approach to achieving a predominantly site-directed mutagenesis has been the introduction of single stranded oligodeoxynucleotides directly into the cell. This techniques has been successfully employed in the yeast Saccharomyces cerevisiae, in which homologous recombination is significantly more active than it is in higher eukaryotes. Moerschell, R. P., et al., 1988, Proc.Natl.Acad.Sci. 85:524-28; Yamamoto, T., et al., 1992, Yeast 8:935-48. However, to date there have been no reports of the successful transformation of mammalian or avian cells by single stranded oligonucleotides.
A relationship between the structure of the target DNA and the rate of homologous recombination in mammalian can be inferred by studies that show that regions of alternating purine and pyrimidine bases, i.e., d(TG).sub.30 .multidot.d(AC).sub.30 !, display an entranced rate of recombination. These effects were demonstrated in studies of non-replicating plasmids in cultured mammalian cells. Wahls, W. P., et al., 1990, Mol. Cell. Biol. 10:785-93. These experiments were not extended to show recombination between an exogenous nucleic acid and the genome of the cell.
Attempts have been made to use RecA, a protein that promotes homologous recombination in the bacteria, E. coli, to promote homologous recombination in eukaryotic cells. However, these attempts have not been clearly successful. For example U.S. Pat. No. 4,950,599 to W. Bertling discloses a very low rate of site-directed mutation and no enhancement in the rate of homologous recombination by use of RecA in eukaryotic cells. Patent publications WO 93/22443 to D. Zarling and E. Sena, and publication 94/04032 to D. C. Gruenert and K. Kunzelmann both purport to correct a genetic defect in a cultured cell line related to cystic fibrosis. These publications disclose primarily experimental data that demonstrate the principle rather than data concerning examples of operative methods. Thus, to introduce polynucleotide/RecA complexes access to the nucleus, Zarling and Gruenert employ cells that were membrane-permeabilized, although such cells are incapable of further growth. Moreover, even when RecA-promoted homologous recombination was asserted to have taken place in intact cells, these publications provide no quantitative estimates of its frequency. Thus, the use of prokaryotic recA has not been convincingly shown to result in a rate homologous recombination in any viable eukaryotic cell significantly greater than the spontaneous rate of homologous recombination.